Forskolin (Fsk), a compound isolated from a plant, has been demonstrated to interact directly with the enzyme adenylyl cyclase in diverse tissues. At least nine different types of adenylyl cyclase have been cloned. The expression of the different types of adenylyl cyclase in a recombinant Sf9 expression system provides an opportunity to study the effects of Fsk on the individual adenylyl cyclases. Reaction of type I adenylyl cyclase with a chemically reactive Fsk derivative (6-NCS-Fsk) causes irreversible inhibition of Fsk activation, suggesting a covalent interaction with6-NCS-Fsk at the activation site. In contrast, the type II enzyme is stimulated by 6-NCS-Fsk, suggesting that there is no reactive nucleophilic group and therefore, no covalent interaction at the activation site of this enzyme subtype. We are using 6-NCS-Fsk to localize the site(s) of forskolin interaction with types I and II adenylyl cyclase and with mixed cytoplasmic domains (C1a/C2a) of type I and type II enzymes, respectively. This research is aimed at probing the molecular properties of adenylyl cyclase, an enzyme which plays a pivotal role in signal transduction. Several of the cytokines that are regulated by the division activate cells through adenylyl cyclase. Characterization of this enzyme and its role in signal transduction enables us to better understand how these cytokines activate cells and helps us prevent potential adverse reactions when these cytokines are used in conjunction with other drugs that modulate adenylyl cyclase activity.